Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical, veterinary, agricultural and horticultural industries. This is particularly the case in the area of diagnostics for human disease conditions. As a greater understanding of genomics is achieved and with the completion or near completion of genome sequencing for a range of animals and mammals, including humans, and a range of microorganisms, there will be greater opportunities to develop diagnostic assays for a wide range of genetic based conditions.
Diagnostic techniques based on nucleic acid hybridization are unparalleled in their ability to identify and quantify genetic material of particular organisms or groups of genetically related organisms. The provision of DNA microfabricated array (micro-array) techniques now allows an “order of magnitude” increase in speed and specificity for this kind of gene-based analysis. For example, reference may be made to Southern (WO 89/10977; U.S. Pat. No. 6,045,270), Chee et al. (U.S. Pat. No. 5,837,832), Cantor et al. (U.S. Pat. No. 6,007,987) and Fodor et al. (U.S. Pat. No. 5,871,928).
Until recently, the nucleic acid probes used in nucleic acid hybridizations were mostly obtained empirically by isolating nucleic acid fragments from targeted organisms or genes. However, it is now possible to design and synthesize nucleic acid probes using data from the international sequence databases (e.g. the GenBank and EMBL databases). These databases of known gene sequences have been increasing tenfold in size every five years for many years and now contain a representative sample of most genes and most major groups of organisms.
Generally, DNA micro-arrays use spots of detector oligonucleotides or probes positioned in arrays on a solid support, typically a glass wafer. The probes are allowed to hybridize with sample nucleic acids, which contain the target nucleic acids and which have been fluorescently labelled. The probes and target nucleic acids of the sample are allowed to hybridize under conditions that only detect exact or almost exact complementarity between the probes and the target nucleic acids. If a target nucleic acid complements and hybridizes to a particular probe in the array, the spot will fluoresce. Recording the fluorescence of the spots enables one to assess which target sequences are present in the nucleic acids mixture.
Sequence information, obtained from native RNA or DNA molecules, is used to determine the sequence of the synthesized oligonucleotide probes and this information is usually stored in computer databases and manipulated using software. Each probe is synthesized so that it contains nucleotides in any order (sequence) that matches a part of a known native nucleotide sequence or the complement of a part of that sequence. Oligonucleotide probes used in conventional arrays are typically 10-25 nucleotides long.
Currently oligonucleotide probes are most commonly used in micro-arrays to identify and quantify the mRNA transcripts from genes. These micro-arrays usually contain probes representing several different target sequences from each gene sequence and these probes are usually chosen to be target specific (i.e. they hybridize with just one target polynucleotide). Thus, these micro-arrays contain many more probes than the number of target polynucleotides they are designed to detect.
Compared to conventional nucleic acid analysis techniques including restriction fragment length polymorphism (RFLP) analysis and the polymerase chain reaction (PCR), DNA micro-arrays provide a facile and rapid means of detecting and measuring the expression of different genes. They have also been used to detect variants of well characterized nucleic acid molecules (i.e. to detect genetic polymorphisms and genotypes). However, despite their promise as tools for diagnosing infectious diseases as well as genetic disorders, the development of micro-arrays for routine diagnosis appears to be slow. This is probably due to the relatively high cost of designing, developing and producing micro-arrays that could detect a larger number of target polynucleotides. New methods and reagents are, therefore, required to realize this promise and the present invention helps to meet that need.
In accordance with the present invention, the inventors have developed an improved assay system which can readily identify changes in nucleotides within a target nucleotide sequence and whether the mutation or polymorphism is present in homozygous or heterozygous form. The assay of the present invention has wide applicability for a range of genetic testing of humans, animals, microorganism and plants. The instant assay has particular utility in microarray-based assay procedures. Furthermore, many individual subjects can be analyzed on the same micro-array, which will allow large-scale genetic testing in a cost-effective manner.